(Schub-)Spannendes aus der Biotechnologie – Blutstrom als Fitness-Training für die Gefäßwand

Mechanische Beanspruchungen verändern bei nahezu jeder Zelle ihre Funktion und ihre Form. Wir interessieren uns besonders für die durch mechanische Beanspruchungen hervorgerufene Effekte im Blutgefäßsystem, dessen Innenseite von den sogenannten Endothelzellen ausgekleidet ist, die eine Permeabilitätsbarriere zwischen Blut und Gewebe darstellen. Durch den Blutstrom sind diese Zellen permanent einer erheblichen mechanischen Beanspruchung ausgesetzt, die nicht nur ihre Form, sondern auch ihre Funktionen wesentlich verändert. Wir haben in unserem Labor einen experimentellen Aufbau entwickelt, mit dem wir erstmalig zeigen konnten, dass laminare Strömungen zu einer Verstärkung der endothelialen Barrierefunktion führen und so vermutlich der Entwicklung der Gefäßverkalkung entgegenwirken. Neben diesen Experimenten wird das neue System auch zur dynamischen Untersuchung der Zellhaftung auf Biomaterialien verwendet.Mechanical loads change the function and morphology of nearly every cell. We are particularly interested in the effects of mechanical loads on the endothelial cells which line the inner surface of blood vessels and control the exchange of water and solutes between blood and tissue (barrier function). These cells are exposed permanently to mechanical forces from the blood stream, which induces changes not only in cell morphology but also in function. We have developed an experimental setup which allows the endothelial barrier function to be measured under defined flow conditions. We have demonstrated for the first time that laminar shear stress enhances the endothelial barrier function, and thus a possible explanation for the anti-arteriosclerotic effect. Importantly, our setup can also be used to dynamically test the adhesion of cells on biomaterials

Segmentierung und Verfolgung für die Migrationsanalyse von Endothelzellen

Endothelzellen bilden eine monozellulare Grenzschicht in Blutgefäßen. Ihre Migration ist ein kritischer Teilschritt bei der Gefäßbildung, zum Beispiel während der Wundheilung. Obwohl bereits eine Reihe der dafür relevanten Mediatoren und pathogenen Determinanten bekannt sind, fehlt bisher eine quantitative Analyse der molekularen Mechanismen der Gefäßbildung und Zellmigration. Voraussetzung dafür sind Verfahren zur automatisierten Bestimmung von Zelltrajektorien in Sequenzen von Mikroskopaufnahmen migrierender Zellverbände. Dazu wurde ein statistisches Modell entwickelt, welches die Segmentierung und Verfolgung von Zellen in Bildsequenzen ermöglicht. Im vorliegenden Beitrag stellen wir dieses Modell vor, diskutieren die sich daraus ergebenden Lern- und Erkennungsalgorithmen und präsentieren erste Resultate.Mechanical loads change the function and morphology of nearly every cell. We are particularly interested in the effects of mechanical loads on the endothelial cells which line the inner surface of blood vessels and control the exchange of water and solutes between blood and tissue (barrier function). These cells are exposed permanently to mechanical forces from the blood stream, which induces changes not only in cell morphology but also in function. We have developed an experimental setup which allows the endothelial barrier function to be measured under defined flow conditions. We have demonstrated for the first time that laminar shear stress enhances the endothelial barrier function, and thus a possible explanation for the anti-arteriosclerotic effect. Importantly, our setup can also be used to dynamically test the adhesion of cells on biomaterials

Caveolin-1 opens endothelial cell junctions by targeting catenins

Aims A fundamental phenomenon in inflammation is the loss of endothelial barrier function, in which the opening of endothelial cell junctions plays a central role. However, the molecular mechanisms that ultimately open the cell junctions are largely unknown. Methods and results Impedance spectroscopy, biochemistry, and morphology were used to investigate the role of caveolin-1 in the regulation of thrombin-induced opening of cell junctions in cultured human and mouse endothelial cells. Here, we demonstrate that the vascular endothelial (VE) cadherin/catenin complex targets caveolin-1 to endothelial cell junctions. Association of caveolin-1 with VE-cadherin/catenin complexes is essential for the barrier function decrease in response to the pro-inflammatory mediator thrombin, which causes a reorganization of the complex in a rope ladder-like pattern accompanied by a loss of junction-associated actin filaments. Mechanistically, we show that in response to thrombin stimulation the protease-activated receptor 1 (PAR-1) causes phosphorylation of caveolin-1, which increasingly associates with β- and γ-catenin. Consequently, the association of β- and γ-catenin with VE-cadherin is weakened, thus allowing junction reorganization and a decrease in barrier function. Thrombin-induced opening of cell junctions is lost in caveolin-1-knockout endothelial cells and after expression of a Y/F-caveolin-1 mutant but is completely reconstituted after expression of wild-type caveolin-1. Conclusion Our results highlight the pivotal role of caveolin-1 in VE-cadherin-mediated cell adhesion via catenins and, in turn, in barrier function regulatio

Coronin 1B Controls Endothelial Actin Dynamics at Cell-Cell Junctions and Is Required for Endothelial Network Assembly

Development and homeostasis of blood vessels critically depend on the regulation of endothelial cell-cell junctions. VE-cadherin (VEcad)-based cell-cell junctions are connected to the actin cytoskeleton and regulated by actin-binding proteins. Coronin 1B (Coro1B) is an actin binding protein that controls actin networks at classical lamellipodia. The role of Coro1B in endothelial cells (ECs) is not fully understood and investigated in this study. Here, we demonstrate that Coro1B is a novel component and regulator of cell-cell junctions in ECs. Immunofluorescence studies show that Coro1B colocalizes with VEcad at cell-cell junctions in monolayers of ECs. Live-cell imaging reveals that Coro1B is recruited to, and operated at actin-driven membrane protrusions at cell-cell junctions. Coro1B is recruited to cell-cell junctions via a mechanism that requires the relaxation of the actomyosin cytoskeleton. By analyzing the Coro1B interactome, we identify integrin-linked kinase (ILK) as new Coro1B-associated protein. Coro1B colocalizes with α-parvin, an interactor of ILK, at the leading edge of lamellipodia protrusions. Functional experiments reveal that depletion of Coro1B causes defects in the actin cytoskeleton and cell-cell junctions. Finally, in matrigel tube network assays, depletion of Coro1B results in reduced network complexity, tube number and tube length. Together, our findings point toward a critical role for Coro1B in the dynamic remodeling of endothelial cell-cell junctions and the assembly of endothelial networks

MAGI1 mediates eNOS activation and NO production in endothelial cells in response to fluid shear stress

Fluid shear stress stimulates endothelial nitric oxide synthase (eNOS) activation and nitric oxide (NO) production through multiple kinases, including protein kinase A (PKA), AMP-activated protein kinase (AMPK), AKT and Ca2+/calmodulin-dependent protein kinase II (CaMKII). Membrane-associated guanylate kinase (MAGUK) with inverted domain structure-1 (MAGI1) is an adaptor protein that stabilizes epithelial and endothelial cell-cell contacts. The aim of this study was to assess the unknown role of endothelial cell MAGI1 in response to fluid shear stress. We show constitutive expression and co-localization of MAGI1 with vascular endothelial cadherin (VE- cadherin) in endothelial cells at cellular junctions under static and laminar flow conditions. Fluid shear stress increases MAGI1 expression. MAGI1 silencing perturbed flow-dependent responses, specifically, Krüppel-like factor 4 (KLF4) expression, endothelial cell alignment, eNOS phosphorylation and NO production. MAGI1 overexpression had opposite effects and induced phosphorylation of PKA, AMPK, and CAMKII. Pharmacological inhibition of PKA and AMPK prevented MAGI1- mediated eNOS phosphorylation. Consistently, MAGI1 silencing and PKA inhibition suppressed the flow-induced NO production. Endothelial cell-specific transgenic expression of MAGI1 induced PKA and eNOS phosphorylation in vivo and increased NO production ex vivo in isolated endothelial cells. In conclusion, we have identified endothelial cell MAGI1 as a previously unrecognized mediator of fluid shear stress- induced and PKA/AMPK dependent eNOS activation and NO productio

(Schub-)Spannendes aus der Biotechnologie – Blutstrom als Fitness-Training für die Gefäßwand

Mechanische Beanspruchungen verändern bei nahezu jeder Zelle ihre Funktion und ihre Form. Wir interessieren uns besonders für die durch mechanische Beanspruchungen hervorgerufene Effekte im Blutgefäßsystem, dessen Innenseite von den sogenannten Endothelzellen ausgekleidet ist, die eine Permeabilitätsbarriere zwischen Blut und Gewebe darstellen. Durch den Blutstrom sind diese Zellen permanent einer erheblichen mechanischen Beanspruchung ausgesetzt, die nicht nur ihre Form, sondern auch ihre Funktionen wesentlich verändert. Wir haben in unserem Labor einen experimentellen Aufbau entwickelt, mit dem wir erstmalig zeigen konnten, dass laminare Strömungen zu einer Verstärkung der endothelialen Barrierefunktion führen und so vermutlich der Entwicklung der Gefäßverkalkung entgegenwirken. Neben diesen Experimenten wird das neue System auch zur dynamischen Untersuchung der Zellhaftung auf Biomaterialien verwendet.Mechanical loads change the function and morphology of nearly every cell. We are particularly interested in the effects of mechanical loads on the endothelial cells which line the inner surface of blood vessels and control the exchange of water and solutes between blood and tissue (barrier function). These cells are exposed permanently to mechanical forces from the blood stream, which induces changes not only in cell morphology but also in function. We have developed an experimental setup which allows the endothelial barrier function to be measured under defined flow conditions. We have demonstrated for the first time that laminar shear stress enhances the endothelial barrier function, and thus a possible explanation for the anti-arteriosclerotic effect. Importantly, our setup can also be used to dynamically test the adhesion of cells on biomaterials

(Schub-)Spannendes aus der Biotechnologie – Blutstrom als Fitness-Training für die Gefäßwand

Mechanische Beanspruchungen verändern bei nahezu jeder Zelle ihre Funktion und ihre Form. Wir interessieren uns besonders für die durch mechanische Beanspruchungen hervorgerufene Effekte im Blutgefäßsystem, dessen Innenseite von den sogenannten Endothelzellen ausgekleidet ist, die eine Permeabilitätsbarriere zwischen Blut und Gewebe darstellen. Durch den Blutstrom sind diese Zellen permanent einer erheblichen mechanischen Beanspruchung ausgesetzt, die nicht nur ihre Form, sondern auch ihre Funktionen wesentlich verändert. Wir haben in unserem Labor einen experimentellen Aufbau entwickelt, mit dem wir erstmalig zeigen konnten, dass laminare Strömungen zu einer Verstärkung der endothelialen Barrierefunktion führen und so vermutlich der Entwicklung der Gefäßverkalkung entgegenwirken. Neben diesen Experimenten wird das neue System auch zur dynamischen Untersuchung der Zellhaftung auf Biomaterialien verwendet.Mechanical loads change the function and morphology of nearly every cell. We are particularly interested in the effects of mechanical loads on the endothelial cells which line the inner surface of blood vessels and control the exchange of water and solutes between blood and tissue (barrier function). These cells are exposed permanently to mechanical forces from the blood stream, which induces changes not only in cell morphology but also in function. We have developed an experimental setup which allows the endothelial barrier function to be measured under defined flow conditions. We have demonstrated for the first time that laminar shear stress enhances the endothelial barrier function, and thus a possible explanation for the anti-arteriosclerotic effect. Importantly, our setup can also be used to dynamically test the adhesion of cells on biomaterials